U.S. patent number 7,521,231 [Application Number 10/522,010] was granted by the patent office on 2009-04-21 for method for preparing engineered tissue.
This patent grant is currently assigned to Organogenesis, Inc.. Invention is credited to Francois Auger, Francois Bergeron, Lucie Germain, Charles Roberge.
United States Patent |
7,521,231 |
Germain , et al. |
April 21, 2009 |
Method for preparing engineered tissue
Abstract
A method for preparing a human or animal tissue by applying a
compressive force to a stack of sheets of living tissue thereby
inducing adjacent layers to fuse or adhere to each other. The force
is applied in direction normal to the surface of the tissue. A
multi-layer tissue produced by the method described above can also
possess at least two different types of sheets and/or consist
essentially of between two and twelve sheets of living tissue. The
method can also be used to prepare a planar tissue that can further
be incorporated in a multi-layer tissue construct. The methods and
tissues described herein are useful for the preparation of
engineered tissues.
Inventors: |
Germain; Lucie
(St.-Augustin-De-Demaures, CA), Auger; Francois
(Sillery, CA), Bergeron; Francois (Sainte-Foy,
CA), Roberge; Charles (Sillery, CA) |
Assignee: |
Organogenesis, Inc. (Canton,
MA)
|
Family
ID: |
30115954 |
Appl.
No.: |
10/522,010 |
Filed: |
July 16, 2003 |
PCT
Filed: |
July 16, 2003 |
PCT No.: |
PCT/CA03/01079 |
371(c)(1),(2),(4) Date: |
November 09, 2005 |
PCT
Pub. No.: |
WO2004/007699 |
PCT
Pub. Date: |
January 22, 2004 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060128010 A1 |
Jun 15, 2006 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60396004 |
Jul 16, 2002 |
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Current U.S.
Class: |
435/325; 424/422;
424/443; 435/366; 435/371; 435/395; 623/1.11; 623/23.72;
623/23.76 |
Current CPC
Class: |
C12N
5/0062 (20130101) |
Current International
Class: |
C12N
5/06 (20060101); C12N 5/08 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 96/31226 |
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Oct 1996 |
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WO |
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WO 97/37613 |
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Oct 1997 |
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WO |
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WO 99/05262 |
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Feb 1999 |
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WO |
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WO 0029553 |
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May 2000 |
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WO |
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WO 00/69355 |
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Nov 2000 |
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WO |
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WO 03/007795 |
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Jan 2003 |
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WO |
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WO 03/045458 |
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Jun 2003 |
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WO |
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Other References
Qing Ye, et al., "Tissue engineering in Cardiovascular surgery: new
approach to develop completely human autologous Tissue," European
Journal of Cardio-Thoracic Surgery, vol. 17, Apr. 2000, pp.
449-454, XP002959353. cited by other .
L'Heureux Nicholas et al., "A Completely Biological
Tissue-Engineered Human Blood Vessel," Journal Fed. of American
Soc. For Experimental Biology, vol. 12, 1998, pp. 47-57.
XP002942845. cited by other .
Byung-Soo Kim et al., "Cyclic Mechanical Strain Mechanical Strain
Regulates the Development of Engineered Smooth Muscle Tissue,"
Nature Biotechnolgy, Nature Publishing, US, vol. 27, No. 10, Oct.
1999, pp. 979-983, XP001145976. cited by other .
Keiichi Kanda et al., "Mechanical Stress Induced Cellular
Orientation and Phenotypic Modulation of 3-D Cultured Smooth Muscle
Cells," Asaio journal, J.B. Lippincott Co., vol. 39, No. 3, Jul. 1,
1993, pp. M686-690. cited by other .
Lopez, C.A. et al., "Peripheral Anchorage of Dermal Equivalents,"
British Journal of Dermatology, vol. 127, No. 4, 1992, pp. 365-371,
XP009021181. cited by other.
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Primary Examiner: Lilling; Herbert J.
Attorney, Agent or Firm: Adamson; Edward J. Dipali; Ravi
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the 371 National Phase of International
Application No. PCT/CA2003/001079, filed Jul. 16, 2003, which was
published in English under PCT Article 21(2) as International
Publication No. WO 2004/007699. This application further claims the
benefit under 35 U.S.C. .sctn. 119 (e) of U.S. provisional
application No. 60/396,004 filed Jul. 16, 2002. All of these
applications are incorporated herein by reference in their
entirety.
Claims
The invention claimed is:
1. A method for preparing a human or animal tissue from at least
one sheet of living tissue, the method comprising the steps of: (a)
arranging said at least one sheet of living tissue to form a
multi-layer stack of living tissue; and (b) applying a compressive
force in a direction normal to the surface of the multi-layer stack
of living tissue with a force-applying means at a pressure and for
an amount of time sufficient to compress layers of tissue together
for inducing adjacent layers of tissue to fuse or adhere to each
other, wherein said force-applying means in step (b) comprises an
adjustable weighted device suitable for applying substantially
evenly-distributed pressure to said multi-layer stack of living
tissue, said weighted device being at least partially permeable to
tissue-culture medium.
2. The method of claim 1, wherein said multi-layer stack is
arranged on a substantially flat support.
3. The method of claim 1, wherein said multi-layer stack of living
tissue in step (a) is formed by superimposing two or more sheets of
living tissue.
4. The method of claim 1, wherein said multi-layer stack of living
tissue is formed by folding a sheet of living tissue upon
itself.
5. The method of claim 1, further comprising a step of anchoring
said multi-layer stack of living tissue with anchoring means before
said step (b) of applying a force, wherein said anchoring means
applies sufficient tension across said multi-layer stack of living
tissue to prevent shrinkage and/or maintain cellular
differentiation and/or induce fiber orientation.
6. The method of claim 5, wherein said anchoring means comprises a
multiplicity of spaced apart weights or ingots arranged
substantially around the perimeter of said multi-layer stack of
living tissue.
7. The method of claim 1, wherein the multi-layered stack of living
tissue in step (a) is formed by rolling a sheet of living tissue on
a tubular support.
8. The method of claim 7, wherein said force-applying means in step
(b) comprises a tissue-culture medium permeable elastic sleeve.
9. The method of claim 1, wherein cells are obtained from said at
least one sheet of living tissue, said at least one sheet of living
tissue is obtained via biopsy.
10. The method of claim 1, wherein said at least one sheet of
living tissue is obtained by culturing cells in vitro.
11. The method of claim 10, wherein said cells are selected from
the group consisting of embryonic stem cells, post-natal stem
cells, adult stem cells, mesenchymal cells, hepatocytes, islet
cells, parenchymal cells, osteoblasts and other cells forming bone
or cartilage, and nerve cells.
12. The method of claim 11, wherein said mesenchymal cells are
selected from the group consisting of fibroblasts, interstitial
cells, endothelial cells, smooth muscle cells, skeletal muscle
cells, myocytes, chrondocytes, adipocytes, fibromyoblasts, and
ectodermal cells.
13. The method of claim 1, wherein said at least one sheet of
living tissue is selected from the group consisting of a skin
tissue, a corneal tissue, a cardiac valve tissue, a connective
tissue and a mesenchymal tissue.
14. A multi-layer tissue made according to the method of claim 1,
wherein said multi-layer tissue comprises at least two different
types of sheets of living tissue.
15. A multi-layer tissue according to claim 14, consisting
essentially of between two sheets and twelve sheets of living
tissue.
16. A multi-layer tissue according to claim 14, consisting
essentially of between three sheets and nine sheets of living
tissue.
17. A multi-layer tissue according to claim 14, wherein said tissue
has a thickness of about 0.01 mm to about 0.5 mm.
18. A multi-layer tissue according to claim 17, wherein said tissue
has a thickness of about 0.03 mm to about 0.45 mm.
Description
FIELD OF THE INVENTION
The present invention relates to engineered tissue. In particular,
the present invention relates to methods for making engineered
tissue from sheets of living tissue.
BACKGROUND OF THE INVENTION
Tissue engineering may be used to recreate tissues and organs for
grafting onto patients. Engineered tissues and organs can also
serve as in vitro models. A variety of tissue engineering
techniques are known, including tissue in-growth, seeding of cells
on artificial or biodegradable scaffolds, and collagen gels. Among
them, a new method of tissue engineering, known as the
"self-assembly" method, has emerged (L'Heureux et al.; Michel et
al.; Pouliot et al.). In the self-assembly method, cells are
induced to secrete and organise an extracellular matrix and thereby
form a sheet of living tissue. The self-assembly method takes
advantage of the fact that fibroblasts can produce a suitable
extracellular matrix when grown in the presence of ascorbic acid.
To create multi-layer tissue constructs, sheets of living tissue
can be stacked upon each other, folded upon themselves, or rolled
on a tubular support.
The development of tissue engineering methods to produce
reconstructed tissues has focused on the optimization of
morphological and histological properties of the reconstructed
tissues. Most tissue engineering research has focused on optimizing
techniques for growing sheets of tissue. However, in many cases, it
would be desirable to make reconstructed tissue comprised of
several layers of tissue and/or layers of more than one type of
tissue. Multi-layer tissue constructs are thicker and therefore
stronger and since multi-layer tissue constructs can comprise more
than one sheet of living tissue, they can be designed to more
closely resemble the tissues that they are intended to replace.
However, in order to create useful multi-layer tissue constructs,
it is essential to be able to fuse adjacent layers of cell tissue
together so that the layers are bonded together as firmly and
reliably as possible and resist separation. If these layers of
tissue are not fused together well, they may separate or come apart
over time, for example, during handling or in the body of a
patient.
Thus, it would be desirable to have a method for preparing
multi-layered engineered tissue constructs with improved fusion
between adjacent layers of tissue.
DESCRIPTION OF BACKGROUND ART
Ye et al. teach that mechanical stress can enhance the synthesis
and secretion of the principal extracellular matrix protein,
collagen, by fibroblasts, thereby increasing the mechanical
strength of the stiffness of reconstructed tissue. Ye et al.
describe a method for applying mechanical stress wherein sheets of
fibroblast cells are mounted on frames to apply tension. However,
the reconstructed tissues produced by this method have
significantly less collagen than does native tissue and therefore
would not be expected to have mechanical properties that resemble
native tissue.
Kanda et al. teach that mechanical stress induces cell orientation
and phenotypic modulation of cultured smooth muscle cells.
L'Heureux et al. describe a method of making a tissue-engineered
blood vessel (TEBV) by wrapping sheets of living tissue around a
tubular support.
Lopez-Valle et al. describe the use of a continuous (as opposed to
punctuated or discontinuous) anchor made of porous glass microfiber
material, the pores of which trap collagen fibers and thereby
induce organization of extracellular matrix and orientation of
cells.
SUMMARY OF THE INVENTION
The current invention provides a method for improving the fusion
between adjacent layers of living tissue in a multi-layered
engineered tissue construct. The current invention further provides
a method for making a sheet of living tissue suitable for use in
preparing a multi-layered engineered tissue construct.
Multi-layered reconstructed tissues produced by this method have
improved fusion between layers of tissue and the layers of tissue
are less likely to separate during subsequent manipulation.
Thus, in one aspect, the invention provides a method for preparing
a human or animal tissue from at least one sheet of living tissue,
the method comprising the steps of: (a) arranging the at least one
sheet of living tissue to form a multi-layer stack of living
tissue; and (b) applying a compressive force in a direction normal
to the surface of the multi-layer stack of living tissue with a
force-applying means at a pressure and for an amount of time
sufficient to compress layers of tissue together for inducing
adjacent layers of tissue to fuse or adhere to each other.
A particular preferred embodiment provides a method for producing a
tissue by forming a multi-layer stack of living tissue arranged on
a substantially flat support. More preferably, the multi-layer
stack is formed by superimposing two or more sheets of living
tissue and/or by folding a sheet of living tissue upon itself.
In a further embodiment, the method comprises another step where
the multi-layer stack is then anchored to a substantially flat
support surface with moveable anchors comprising weights or ingots,
wherein the anchors are of a suitable weight for (1) applying
sufficient tension across the sheet of living tissue to prevent
shrinkage and/or maintain cellular differentiation and/or induce
orientation of cells in at least one sheet of living tissue and (2)
allowing contraction of at least one sheet of living tissue once a
predetermined threshold of tension is exceeded across the sheet of
living tissue. A force is then applied normal to the surface of the
layers of tissue by way of a weighted device suitable for applying
evenly distributed pressure to the surface of the multi-layer stack
of tissue, the weighted device being at least partially permeable
to culture medium, for inducing adjacent layers of tissue to fuse
to each other.
In a preferred embodiment, the force-applying means in step (b) of
the method comprises a weight device suitable for applying
substantially evenly-distributed pressure to said multi-layer stack
of living tissue, the weight device being at least partially
permeable to tissue-culture medium.
In a further embodiment, the multi-layer stack of step (a) of the
method is formed by rolling a sheet of living tissue on a tubular
support and, more preferably, the force-applying means of step (b)
comprises a tissue-culture medium permeable elastic sleeve.
There are many different types of sheets of living tissues that can
be used with the method described herein. In an embodiment, the
method utilizes a biopsy as a sheet of living tissue. In another
embodiment, the method utilizes cells cultured in vitro as a sheet
of living tissue. More preferably, the cells are embryonic stem
cells, post-natal stem cells, adult stem cells, mesenchymal cells,
hepatocytes, Islet cells, parenchymal cells, osteoblasts and other
cells forming bone or cartilage, and nerve cells. The mesenchymal
cells can either be fibroblasts, interstitial cells, endothelial
cells, smooth muscle cells, skeletal muscle cells, myocytes,
chrondocytes, adipocytes, fibromyoblasts, or ectodermal cells. In a
further embodiment, the sheet of living tissue can also be a skin
tissue, a corneal tissue, a cardiac valve tissue, a connective
tissue and/or a mesenchymal tissue.
In another aspect, the invention also provides a multi-layer tissue
made according to the method described herein, wherein the
multi-layer tissue comprises at least two different types of sheets
of living tissue. In a preferred embodiment, the tissue consists
essentially of between two and twelve sheets, more preferably of
between three and nine sheets. In another embodiment, the tissue
has a thickness of between about 0.01 mm to about 0.5 mm, more
preferably of between about 0.03 mm to about 0.45 mm.
In a further aspect, the invention provides a method for preparing
a planar human or animal tissue suitable for use in making a
multi-layer tissue construct from at least one sheet of living
tissue, the method comprising the steps of: (a) arranging said at
least one sheet of living tissue on a substantially flat support
surface; and (b) anchoring said at least one sheet of living tissue
to the support surface with an adjustable anchor-means comprised of
a multiplicity of spaced apart anchors, wherein the anchors are
suitable for (1) applying sufficient tension across the sheet of
living tissue to prevent shrinkage and/or maintain cellular
differentiation and/or induce orientation of cells in said at least
one sheet of living tissue and (2) allowing contraction of said at
least one sheet of living tissue once a predetermined threshold of
tension is exceeded across the sheet of living tissue.
Preferably, the adjustable anchor-means is comprised of discrete
moveable anchors such as weights or ingots. Alternatively, the
adjustable anchor means may also comprise a moveable frame or a
multiplicity of moveable anchors mounted on a frame.
In a particular preferred embodiment, a planar construct can be
made by forming a multi-layer stack of living tissue by
superimposing two or more sheets of living tissue or by folding a
sheet of living tissue upon itself. The multi-layer stack is then
anchored to a substantially flat support surface with moveable
anchors comprising weights or ingots, wherein the anchors are of a
suitable weight for (1) applying sufficient tension across the
sheet of living tissue to prevent shrinkage and/or maintain
cellular differentiation and/or induce orientation of cells in at
least one sheet of living tissue and (2) allowing contraction of at
least one sheet of living tissue once a predetermined threshold of
tension is exceeded across the sheet of living tissue. A force is
then applied normal to the surface of the layers of tissue by way
of a weighted device suitable for applying evenly distributed
pressure to the surface of the multi-layer stack of tissue, the
weighted device being at least partially permeable to culture
medium, for inducing adjacent layers of tissue to fuse to each
other.
In a further embodiment, the sheet of living tissue used to prepare
the planar construct is obtained by culturing cells in vitro.
In yet another aspect, the invention provides a planar multi-layer
tissue consisting essentially of between two to twelve sheets of
living tissue obtained by the method described herein.
In another particular embodiment, a tubular construct can be made
by forming a multi-layer stack of living tissue by rolling a sheet
of living tissue onto itself, for example with the aid of a tubular
support. A culture medium permeable elastic sleeve can be used to
compress the layers of tissue in the multi-layer stack of living
tissue together for inducing the adjacent layers of tissue to fuse
to each other.
Other embodiments and advantages of the invention will become
apparent from the detailed description to follow, together with the
accompanying drawings.
All publications, figures, patents and patent applications cited
herein are hereby expressly incorporated by reference in their
entirety for all purposes to the same extent as if each was so
individually denoted.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a preferred method of making a planar tissue.
(A) A first sheet of living tissue (1) is arranged on a
substantially flat support surface and anchored peripherally with
weights or ingots (2); a second sheet of tissue (3) is superimposed
on the first sheet of tissue, and the weights or ingots (2) are
moved, one-by-one, and placed on the second sheet, thereby
anchoring the superimposed sheets. (B) A sponge (4) that has been
cut to fit within the ingots is placed on top of the multi-layer
tissue construct (5) so formed; and spaced-apart weights (6) are
placed on the sponge.
FIG. 2 illustrates a preferred method of compressing a tubular
tissue construct. An elastic sleeve (7) is placed around a hollow
tube (8) using its tapered end (9). The hollow tube (8) is larger
than a tissue construct (10) which has been rolled around a mandrel
(11). The hollow tube (8) is then placed around the tissue
construct (10). The elastic sleeve (7) is transferred from the
hollow tube (8) to the tissue construct (10) by gently displacing
the tube (8) in one direction and the tissue construct (10) in the
opposite direction.
FIG. 3 is a microscopic view of the tissue made according to the
method of the invention, after maturation. The tissue is assembled
from nine sheets of living tissue containing fibroblasts and
extracellular matrix constituents. Magnification 20.times., scale
bar 50 .mu.m.
FIG. 4 graphs results of cyclic stress-strain test on a three-layer
tissue construct made as described in Example 1.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In accordance with the present invention, it has been found that
application of a compressive force normal to the plane of a sheet
of tissue enhances fusion between adjacent layers of sheets of
tissue. Compression improves contact between layers of tissue and
encourages fusion of the layers of tissue. By way of example, a
weighted device (for example, a sponge upon which spaced apart
weights are placed) may be applied to a stack of two or more
superimposed planar sheets of tissue, thereby applying a force
normal to the plane of the sheets of tissue (see FIG. 1B). In
another example, a suitably sized elastic sleeve can be fitted over
a multi-layer stack of tissue made by rolling at least one sheet of
tissue onto a tubular support, whereby the elastic sleeve applies
pressure normal to the two-dimensional plane of the sheet of tissue
(see FIG. 2).
Preferably, the compressive force or pressure is applied evenly on
the entire tissue surface. Therefore, it is preferable that a
device adapted to the shape of the tissue be used to induce the
fusion. The amount of pressure applied to the surface of the tissue
stack can be adjusted according to the needs of the engineered
tissue. This pressure is applied for a period of time sufficient to
allow the complete fusion of the tissue layers, preferably between
24 hours to 7 days.
It is also preferable that the device used to induce pressure to
the surface of the tissue be permeable to culture media in order to
allow the nutrition of the living cells. An acceptable way to
generate this pressure on a flat tissue is to lay a semi-rigid
sponge on the top of the tissue stack. Additional weight (for
example, one or more solid ingots) can be distributed on top of the
sponge to obtain the desired amount of pressure on the tissue (see
FIG. 1B). Of course, any other system using mechanical or hydraulic
pressure could be used to provide this compression.
In the example of a tubular or cylindrical construct, the
compressing device should preferably apply equal pressure on the
external surface of the construct. In this particular case, a good
way to compress the tissue is to apply an elastic and permeable
sleeve around the construct (see FIG. 2). The size and the
elasticity of the sleeve can be adjusted to give the appropriate
pressure for a given tissue.
To wrap the sleeve around the tissue and remove it without damage,
an installation device may be used (see FIG. 2). The elastic sleeve
(7) is mounted on a rigid hollow tube (8) using the tapered end of
the tube (9). The tube, having an internal diameter slightly larger
than the construct, is then passed over the tubular tissue (10)
rolled around a mandrel (11). The end of the sleeve is then
anchored to the mandrel and the hollow tube is carefully removed by
pulling from the opposite side, gently depositing the sleeve on the
tissue. To remove the sleeve without damaging the tissue, it may be
for example carefully cut or unsewn.
It is known that mechanical stress may be used to induce cellular
orientation and phenotypic modulation of cultured smooth muscle
cells (Kanda et al.; Germain et al.). Thus, appropriate forces may
be applied to maturating tissue in order to induce fiber
orientation. Such forces may also prevent shrinkage and maintain
the desired cell differentiation.
As an example, it has been shown that a continuous anchor, such as
a frame or a ring of glass microfiber that circumscribes or
encircles a tissue, may be used to induce cellular orientation (Ye
et al.; Kanda et al.). The induction of cell orientation is thought
to occur because the continuous anchor mechanically restricts the
spontaneous contraction of the maturing cultured tissue, thereby
creating a mechanical stress or tension across the tissue that
induces cell orientation.
The underlying mechanism of the orientation response has not been
well elucidated (Kanda et al.). However, when a continuous anchor
is used, the tension across the tissue continues to build as the
tissue matures and can reach levels that are detrimental to the
health of the cells in the tissue, reducing viability of cells
contained in the tissue and thereby producing an inferior tissue
construct. Therefore, a continuous anchor, such as a rigid frame,
may not be suitable for use with some tissue types, i.e. those
tissues that can create a lot of tension as they mature.
The current invention provides an improved method of anchoring
maturing cultured tissues, the method comprising an adjustable
anchor means, preferably comprising a multiplicity of spaced apart
anchors (such as moveable weights or ingots), wherein the anchors
are suitable for (1) applying sufficient tension across the sheet
of living tissue to prevent shrinkage and/or maintain cellular
differentiation and/or induce orientation of cells in at least one
sheet of living tissue and (2) allowing contraction of at least one
sheet of living tissue once a predetermined threshold of tension is
exceeded across the sheet of living tissue (for an illustration see
FIG. 1A).
The anchor means is "adjustable" in that once the tissue has built
up a tension higher than the maximum tension that can be held by
the anchors (i.e. weights or ingots), the tissue can spontaneously
contract and the anchors will be pulled along with the contracting
tissue. Thus, the tension across the tissue cannot continue to
build up when an adjustable anchor means as described is employed.
The maximum tension that can build up across the tissue can be
controlled by choosing suitable anchors (for example weights or
ingots of a certain weight and number, or an adjustable frame that
is designed to move in response to a certain tension or force).
Thus, it is possible to optimize the amount of tension for any
given tissue, for example, to enhance viability of cells in the
tissue.
Anchors according to the current invention may be "discontinuous"
or "punctual". A "discontinuous" or "punctual anchor" is a device
that anchors a tissue substantially at a point in space. In
contrast, in the context of the present invention, the term
"continuous anchor" refers to a device for securing a tissue around
its entire perimeter (such as described by Lopez-Valle et al.).
The anchors of the present invention may be "moveable" in that they
can easily be placed on a sheet of tissue or removed therefrom.
For making planar sheets of living tissue for use in making
multi-layer tissue constructs, it is preferred that anchors are
arranged to form a closed perimeter near the edge of a sheet of
tissue. This geometry induces cells and extracellular matrix fibers
in the sheet of tissue to orient in the two dimensions of the plane
of the sheet of tissue. This orientation of cells and extracellular
matrix may be beneficial for fusion of adjacent layers of sheets of
tissue and may also improve certain functional properties of the
tissue. For example, in FIG. 1A, a first sheet of living tissue (1)
is disposed on a flat surface and ingots (2) keep the first sheet
in place. A second sheet of living tissue (3) is placed on top of
the first sheet. Ingots are displaced from the first sheet and are
arranged on top of the second sheet to anchor the stack of sheets.
The ingots are arranged to follow the perimeter of the stack of
living sheets. The ingots also provide a discontinuous mechanical
force to the living sheets allowing cellular differentiation and
contraction. The process may be repeated to obtain a multi-layer
tissue construct.
The current invention provides the use of a multiplicity of spaced
apart weights or ingots as anchors for applying mechanical force to
tissue in a punctuated or discontinuous manner along the edge of
the sheet of living tissue. If the weights or ingots are arranged
very close to each other or so as to contact each other, they may
displace each other somewhat when the tissue contracts. The amount
and direction of mechanical force applied to a sheet of tissue can
be controlled by varying the number, weight and position of the
weights or ingots. Hence, it is possible to optimize or fine-tune
the mechanical force conditions for any particular size or type of
tissue.
The current invention is in contrast to the continuous anchor made
of porous glass microfiber material described by Lopez-Valle et al.
A porous continuous anchor like that described in Lopez-Valle et
al. is not easily moved, removed or adjusted, and as a result, does
not provide one with the ability to fine-tune the application of
mechanical force to a tissue.
Weights or ingots for use as anchors according to the current
invention may be made from any material that does not interfere
with the development or differentiation of cells in the sheet of
living tissue, such as stainless steel. Magnets or metal ingots
coated with Teflon.TM. or any polymer material known in the art to
be compatible with tissue culture may also be used. Suitable weight
values for the weights or ingots for use with a tissue type can be
determined empirically. Preferably, weights are chosen so that cell
orientation and/or differentiation are induced.
The foregoing technique of using adjustable/moveable anchors and
compression to fuse tissues together also may be used for producing
three-dimensional tissue constructs.
Preparation of Sheets of Living Tissue
Sheets of living tissue for use in making multi-layered
reconstructed tissue in accordance with the current invention may
be obtained from biopsy or may be made using any known techniques.
In the case where sheets of living tissue of mesenchymal origin are
prepared using tissue engineering techniques, a preferred method is
the self-assembly approach, which allows normal cell-cell and
cell-extracellular matrix interactions. In addition, the
self-assembly approach allows the secretion of important natural
growth factors and cytokines, and the formation of a mature
connective tissue necessary for functionality of the tissue and for
the cells in the tissue to remain metabolically active and undergo
normal mitosis.
The subsections below describe preparation and use of human
engineered tissue in vitro. However, the invention is not limited
to human engineered tissue and extends to animal tissue and
engineered tissue with transformed (human and non-human) cells as
well.
Cell Source
A variety of cells can be used in the human engineered tissue of
the present invention. Preferred cell types include embryonic stem
cells, amniotic fluid cells, post-natal stem cells, adult stem
cells, mesenchymal cells, especially fibroblasts, interstitial
cells, endothelial cells, smooth or skeletal muscle cells, myocytes
(muscle stem cells), chrondocytes, adipocytes, fibromyoblasts, and
ectodermal cells, including ductile and skill cells, hepatocytes,
Islet cells, cells present in the intestine and other parenchymal
cells, osteoblasts and other cells forming bone or cartilage, bone
marrow cells and blood cells. In some cases it may also be
desirable to include nerve cells.
Cells can also be genetically engineered to provide additional or
normal function. Methods for genetically engineering cells with
retroviral vectors, polyethylene glycol, and other methods known to
those skilled in the art can be used.
Cells may be autologous, allogeneic or xenogeneic, however
autologous or allogeneic cells are preferred. Immunologically inert
cells, such as embryonic or fetal cells, stem cells, and cells
genetically engineered to avoid the need for immunosuppression may
also be used. Methods and drugs for immunosuppression are known to
those skilled in the art of transplantation.
In some embodiments, cells are obtained by biopsy and dissociated
using standard techniques, such as digestion with a collagenase,
trypsin or other protease solution. For example, the dermal layer
of a skin biopsy can be digested with collagenase according to the
method of Germain and Auger. After the digestion of the dermal
fragments, mesenchymal cells are harvested following centrifugation
and expanded in cell culture media. All cell cultures are used
between their fourth and eight passages, and kept incubated at
37.degree. C. and 8% CO.sub.2. Cells can be easily obtained through
a biopsy anywhere in the body, for example, skeletal muscle
biopsies can be obtained easily from the arm, forearm, or lower
extremities, and smooth muscle can be obtained from the area
adjacent the subcutaneous tissue throughout the body. The biopsy
can be readily obtained with the use of a biopsy needle, a rapid
action needle which makes the procedure extremely simple and almost
painless. Cells may also be procured from, for example, blood
vessels, blood, such as umbilical cord blood, valves and discarded
tissues, such as foreskins and tissue obtained during esthetic or
cosmetic surgical procedures.
Fibroblasts, such as dermal fibroblasts or adventitial fibroblasts,
may be used. Fibroblasts are easily available, and they are the
primary collagen secreting cells in connective tissues. Dermal
fibroblasts are typically harvested from normal adult skin
specimens removed during reductive breast surgery, or from neonatal
foreskin. The potential of human fibroblasts for cardiovascular
application is enormous for both allogeneic and autologous grafts
since cells contained in one square-inch of foreskin can be used to
grow many acres of tissue.
Preparation of a Sheet of Living Tissue
The engineered tissue of the present invention is formed from at
least one sheet of living tissue. Each sheet of living tissue is
comprised of cells and an endogenous extracellular matrix. The
extracellular matrix is secreted by cells, such as mesenchymal
cells, embryonic stem cells or adult stem cells, to name a few.
When mesenchymal cells, such as dermal fibroblasts, are cultured in
a planar culture substratum using L-ascorbic acid or a phosphate
derivative of L-ascorbic acid (e.g. Asc 2-P), serum, and growth
factors, they show an abundant synthesis of extracellular matrix
proteins. This creates the basis of the endogenous extracellular
matrix. L-ascorbic acid plays an important role since it is a
cofactor for the hydroxylation of proline and lysine residues in
collagen (Hata and Senoo), and also it increases both the rate of
transcription of procollagen genes and stability of procollagen
mRNA (Tajima and Pinnell). The extracellular material is comprised
of different proteins, such as collagen type I, other collagen
types (fibrillar and non-fibrillar), elastin, fibrillin,
glycosaminoglycans (such as decorin), growth factors, and
glycoproteins, to name a few.
In the context of the present invention, the resulting living
tissue formed from the cells and the extracellular matrix as
described above is called a "sheet of living tissue".
An exemplary embodiment of methodology for generating such sheets
of living tissue is described in U.S. Pat. No. 5,618,718 by Auger
et al. In summary, Auger et al. describe that dermal fibroblasts,
at a concentration equivalent to 10.sup.4 cells/cm.sup.2, are
plated into 75 cm.sup.2 sterile Petri dishes. Cell medium is
supplemented with a 3:1 DMEM and Ham's F12 modified medium, fetal
bovine serum, penicillin and gentamicin, and with an ascorbic acid
solution. For example, a final ascorbic acid solution between
50-100 .mu.g/ml can be used every other day. Culture conditions are
kept at 92% air and 8% CO.sub.2 at full humidity. Culture time is
approximately three weeks. At the end of the maturation time, the
sheet of living tissue spontaneously detaches from the
substratum.
It can be appreciated that a variety of methods can be used to
prepare the sheets of living tissue (e.g. Auger et al.; Ye et al.;
L'Heureux et al.; Michel et al.; Pouliot et al.) and the present
invention is not limited in scope by using one particular shape
(i.e. thickness and size), cell type, origin, age, maturation time,
component concentration, and culture conditions to generate the
sheet of living tissue.
Preparation of Engineered Tissue
The engineered tissue of the present invention is formed from
superimposing a plurality of individual sheets of living tissue. In
an embodiment, the number of sheets varies between two and twelve.
As described above, the sheets of living tissue are comprised of an
extracellular matrix secreted by cells, such as mesenchymal cells.
The extracellular matrix is produced with many in vivo-like
properties including supramolecular organization of collagen.
Collagen is not only processed, but is also cross-linked
efficiently and the collagen fibrils are assembled into bundles.
When the sheet is layered upon itself, for example by folding or
wrapping, or a plurality of sheets are stacked or superimposed, a
three-dimensional construct having desired structural
characteristics is formed in culture.
In some embodiments, the sheets of living tissue are stacked in a
cell culture dish, either directly superimposed or in an
overlapping fashion. By overlapping, tissues of various shapes may
be formed. For example, rectangular sheets of living tissue may be
arranged in an overlapping fashion to create a circular layered
tissue construct. Or, irregularly shaped cell cultured sheets may
be stacked in a manner to form a regularly shaped tissue. In
addition, the individual sheets may be stacked in the same
orientation or the orientation of the sheets may be varied to
create specific effects in the resulting tissue.
Alternatively or in addition, one or more sheets of living tissue
can be folded to form a multitude of layers. For example, one sheet
may be folded upon itself in an accordion-type fashion or in
repeated halves to superimpose portions of the sheet upon itself.
Or, two or more sheets may be stacked to form a multi-layer stack
of tissue, which multi-layer stack of tissue may then be folded
upon itself to create even more variety of layering.
Alternatively or in addition, a wrapping technique, such as
wrapping a sheet around itself in the style of a cinnamon roll, can
be used to create a multi-layer stack of tissue. It is possible to
combine different/many techniques, for example by first creating a
multi-layer stack of tissue by (1) layering more than one sheet of
tissue and/or (2) folding a sheet or a multi-layer stack of sheets
of tissue on itself, then wrapping the multi-layer stack of (1) or
(2) around itself.
When layering, the sheets of living tissue are held together by
surface adhesion between the sheets. Any number of sheets of living
tissue may be used, preferably five or more, more preferably seven
or more, and more preferably, nine, ten or eleven or more. The
sheets are delicately handled with forceps and superimposed or
otherwise assembled to form the human engineered tissue construct.
By maintaining this construct in culture medium supplemented with
ascorbic acid under conditions similar to those described in Huynh
et al., the sheets of living tissue will fuse together to form a
human engineered tissue resembling the corresponding mature tissue
(FIG. 3).
For some applications, it is preferred that the resulting
reconstructed tissue comprises more than one type of sheet of
living tissue. For example, a reconstructed tissue suitable for use
as a skin graft may comprise sheets of a dermal equivalent and
epidermal equivalent. A reconstructed tissue suitable for use as a
corneal graft should comprises the following layers: an epithelial
equivalent; a stromal equivalent; and endothelial equivalent.
Maturation time of the construct will depend on the nature of the
tissue and the specific mechanical properties desired. For example,
it has been found that mechanical strength of certain tissue
constructs plateau after seven weeks of maturation (L'Heureux et
al.). For any given tissue construct, the maturation time necessary
to obtain optimal functionality may be readily determined using
routine methods known in the art.
Generally, the engineered tissue is thin enough to allow oxygen
delivery through its surfaces to maintain metabolic needs yet thick
enough to provide desired functionality. The current embodiments of
the engineered tissue of the present invention are avascular,
wherein the tissue does not include a microvasculature to deliver
oxygenated blood to the tissue. Therefore, the tissue relies on
oxygen diffusion from its surfaces to sustain the tissue. Due to
oxygen diffusion limitations, the tissue thickness is currently an
important consideration. (Weind et al.). The thickness of the
engineered tissue may be controlled by choosing the number of
sheets of living tissue used. The engineered tissue may have a
thickness ranging from approximately 0.01 mm to 0.5 mm; more
preferably between 0.03 mm to 0.45 mm. Preferred thickness will
vary depending on the tissue type, intended function of the
engineered tissue and the type of cells used.
If required, mature tissue constructs may be cut into a desired
shape using any suitable method, such as die cutting and template
cutting.
In an embodiment, cells from many different species and/or
transformed cells can be used. Since it is contemplated that many
applications of engineered tissue will concern treatment of human
patients, human engineered tissue is especially preferred.
Preconditioning
If desired, the tissue-engineered construct may be preconditioned
to reduce shrinkage, for example as described in the US application
by Lafrance et al.
Although various embodiments of the invention are disclosed herein,
many adaptations and modifications may be made within the scope of
the invention in accordance with the common general knowledge of
those skilled in this art. Such modifications include the
substitution of known equivalents for any aspect of the invention
in order to achieve the same result in substantially the same way.
Numeric ranges are inclusive of the numbers defining the range. In
the claims, the word "comprising" is used as an open-ended term,
substantially equivalent to the phrase "including, but not limited
to". The following examples are illustrative of various aspects of
the invention, and do not limit the broad aspects of the invention
as disclosed herein.
EXAMPLE 1
Preparation of a Reconstructed Multi-Layered Human Tissue Construct
from Sheets of Living Tissue Containing Fibroblasts and
Extracellular Matrix Constituents.
The following example describes a method for preparing a
reconstructed multi-layered human tissue construct from sheets of
living tissue containing fibroblasts and extracellular matrix
constituents according to the present invention (see FIG. 1 for
illustration of the method). All of the procedures described below
are done under sterile conditions, preferably using a sterile flow
hood. It can be appreciated that a variety of methods can be used
to prepare the multi-layered tissue construct and this example is
not intended to limit the scope of this invention to the number of
sheets of tissue superimposed, to one particular shape (i.e.,
thickness and size), cell type, origin, age, maturation time,
component concentration, and culture conditions to generate the
multi-layered human tissue construct. One skilled in the art can
readily appreciate that various modifications can be made to the
method without departing from the scope and spirit of the
invention.
In this example, to produce a sheet of living tissue, 750,000
viable sub-cultured human skin fibroblasts are seeded in a standard
75 cm.sup.2 sterile petri dish for a final seeding density of
10.sup.4 cells/cm.sup.2. Cells are fed with culture medium (DME
containing 10% fetal calf serum (FCS), 100 IU/ml penicillin and 25
.mu.g/ml gentamicin), and cultivated for 4 weeks to form sheets
that can be manipulated. The culture medium is changed three times
per week. A freshly prepared solution of ascorbic acid is added
each time the medium is changed to obtain a final concentration of
50 .mu.g/ml of ascorbic acid. During culture, cells are kept in a
humidified atmosphere (92% air and 8% CO.sub.2).
After the sheets of tissue are formed, they are peeled from the
dishes, and three separate sheets of living tissue are superimposed
using the following technique. A first sheet of living tissue is
put into a petri dish and culture media is added over the sheet to
keep it wet and to help to spread it. Stainless steel ingots
(approximately 1 mm.times.2 mm.times.8 mm) are placed around the
tissue sheet perimeter to keep the tissue sheet anchored and
stretched to its maximal area on the surface of the petri dish.
Another sheet of tissue is then placed on top of the first sheet of
tissue. One by one, the ingots are carefully pushed aside from the
first sheet and other ingots were placed around the tissue sheet
perimeter of the second layer, spreading it over the first sheet of
tissue. These steps were repeated to obtain a three-layered tissue
construct.
A semi-rigid sponge permeable to the culture media is then cut to
fit the size of the tissue construct between the ingots and applied
to the surface of the construct (see FIG. 1B). The sponge should
closely fit the perimeter delimited by the ingots, but not overlap
or exceed it. Ingots are then evenly distributed on the sponge
surface to put some weight on it (in this case, 11 g/40 cm.sup.2
[0.275 g/cm.sup.2]). The sponge as well as the ingots are removed
24 hours to 7 days following the stacking.
Seven days after the stacking of the sheets of tissue, three
three-layered tissue constructs were superimposed to form the final
nine-layered tissue construct using the same technique as described
above. The constructs were further incubated for up to 8 weeks and
culture medium refreshed 3 times a week. The tissue constructs are
then ready for shipment processing.
EXAMPLE 2
Microscopic Analysis of the Tissue Construct
The tissue construct is prepared according to the procedure
described in Example 1. In this example, the tissue construct is
assembled from nine sheets of living tissue.
Biopsies of the living tissue construct are first fixed in
Bouin's.TM. solution. Cross-sections of the fixed tissue are
embedded in paraffin. The cross-sections are stained with Masson's
trichrome. Microscopic observations are done on a Nikon TS100.TM.
microscope at 20.times. magnification.
FIG. 3 shows a microscopic cross-section of the tissue construct
obtained after the stacking and maturation of 9 sheets of living
tissue containing fibroblasts and extracellular matrix
constituents. This light microscopy demonstrates a tissue construct
resembling that of a native tissue with dense extracellular matrix.
In addition, the 9 superimposed sheets of living tissue have fused
together to form one single tissue construct.
EXAMPLE 3
Biomechanical Properties of the Tissue Construct
Mechanical properties of the tissue are determined by simple
tensile tests and cyclic tensile tests. These tests are performed
using a Tytron.TM. 250 MicroForce Testing System, (MTS Systems
Corporation). This machine allows the loading and unloading of the
tissue at different speed rates, and makes data acquisition of the
stress and the deformation applied to the tissue. Both tests are
made on 7.9 mm width tissue slices, for a total of three slices per
tissue. Traction speed is set to 1 mm/s for both tensile and cyclic
tests.
A simple tensile test consists in stretching the tissue until the
load becomes high enough to break it. It allows the measure of the
modulus of elasticity and the ultimate tensile strength of the
tissue. These two values give the relative stiffness and resistance
of the tissue.
Cyclic tensile tests allow determination of the percentage of
plastic deformation of the tissue following a stretch. The
percentage of plastic deformation evaluates the capacity of a
tissue to recover its original shape after a load is applied to it.
The cyclic tensile test is performed by stretching the tissue until
10% of the ultimate tensile strength of the tissue is reached. Then
the traction is stopped and the load removed from the tissue at the
same rate it was applied previously. This result gives the amount
of irreversible deformation the tissue had to endure while it was
stretched.
FIG. 4 graphs cyclic stress-strain test on a mature three-layer
tissue constructs made as described in Example 1. The tissue
construct is resistant to tensile stress. It also has the capacity
to recover its original shape after a 10% strain.
Throughout this application, various references are referred to
describe more fully the state of the art to which this invention
pertains. The disclosures of these references are hereby
incorporated by reference in their entirety into the present
disclosure for all purposes.
REFERENCES
Auger et al. U.S. Pat. No. 5,618,718 issued Apr. 8, 1997. Germain
and Auger. "Tissue engineered biomaterials: biological and
mechanical characteristics", In: Wise, Trantolo, et al. editors:
"Encyclopedic handbook of biomaterials and bioengineering", NY,
N.Y.: Marcel Dekker Inc., 1995, pp. 699-734. Germain et al. Patent
application WO 03/045458, published Jun. 5, 2003. Huynh et al. U.S.
Pat. No. 5,928,281 issued Jul. 27, 1999. Hata and Senoo. J Cell
Physiol. (1989) 138,8-16. Kanda et al. ASAI0 Journal (1993) 39,
M686-90. Lafrance et al. US application Serial No. 20030027332
published Feb. 6, 2003. L'Heureux et al. The FASEB Journal (1998)
12, 47-56. Lopez-Valle et al. British Journal of Dermatology (1992)
127, 365-371. Michel et al. In Vitro Cell Dev Biol Anim (1999) 35,
318-26. Pouliot et al. Transplantation (2002) 73, 1751-7. Tajima
and Pinnell. Biochem Biophys Res Commun. (1982) 106, 632-7. Weind
et al. J Thorac Cardiovasc Surg (2002) 123, 333-40. Ye et al.
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